The State of Cell Blocks and Ancillary Testing

Transcription

1 Special Section Molecular Cytopathology: Update on Molecular and Cytogenetic Applications of Cytologic Specimens Part II The State of Cell Blocks and Ancillary Testing Cell blocks are an integral part of cytology, but their utility is recognized probably more now than ever before, largely owing to the significant role they play in ancillary testing, particularly molecular diagnostics. Modifications to improve the cell block method initially introduced more than a century ago have been made over the years. Though their value is acknowledged and they are widely used across laboratories, cell block preparations are not standardized and results of ancillary testing performed on them are inconsistent. This article reviews the state of cell blocks summarizes the more common, currently available and used methods and their corresponding advantages and shortcomings, outlines the role of alternative techniques (eg, smears), and proposes methods to optimize results. (Arch Pathol Lab Med. 2016;140: ; doi: /arpa RA) CELL BLOCKS: ROLE IN DIAGNOSTIC CYTOPATHOLOGY AND BEYOND The traditional cytology preparation consists of a smear, which is routinely used but often in combination with alternative and more newly introduced technique(s), including cell blocks (ie, cohesive cell pellets formed in a cytology laboratory and subsequently embedded in paraffin and processed like surgical specimens to generate hematoxylin-eosin [H&E] slides) and liquid-based cytology (LBC), respectively. LBC yields a thin layer of cells dispersed over a fixed area, generally by using automated systems (eg, ThinPrep, Hologic Corporation, Marlborough, Massachusetts; and SurePath, Becton Dickinson, Franklin Lakes, New Jersey) that standardize slide preparation, transportation, and fixation (ie, alcohol based). Each preparation, stain (eg, Diff- Accepted for publication June 7, Published as an Early Online Release August 24, From the Department of Pathology, Columbia University Medical Center, New York, New York. Dr Saqi has served as a consultant for Boston Scientific, Focus Pointe Global, and Navigant Consulting. Dr Saqi is a co-inventor on US patent application , Medical Apparatus and Method for Collecting Biological Samples, published October 15, A device based upon this patent, XCellent, has been licensed. Reprints: Anjali Saqi, MD, MBA, Department of Pathology, Columbia University Medical Center, 630 W 168th St, VC14-215, New York, NY ( aas177@cumc.columbia.edu). Past, Present, and Future Anjali Saqi, MD, MBA Quik, Papanicolaou, H&E), and fixative (eg, alcohol based, formalin, saline, among others) offers its unique advantage(s) and often complements the others. Selection of a specific combination generally varies with specimen type, cellularity, (presumed) diagnosis, and laboratory preference. For example, cell blocks are typically prepared from fine-needle aspirations (FNAs), particularly deep-seated lesions and some exfoliative specimens, such as effusions and bronchial washings. 1 3 More commonly, cell blocks from FNAs and exfoliative specimens are not the only preparation but are made in conjunction with smear(s) and/or LBC. Cell blocks, first implemented more than a century ago 4 for ascites fluids and more widely accepted in practice in 1947, 5 have many advantages. They maintain architecture, which most closely resembles that seen on surgical specimens. Namely, small tissue fragments appear as mini biopsies, 2 which are useful for diagnosis, pattern recognition/subclassification, 6 and identification of features (eg, intercellular bridges in squamous cells, unsuspected parathyroid tissue 7 ) that may otherwise be difficult to appreciate on non cell block cytology preparations. For those not accustomed to or lacking proficiency in examining smears or LBC routinely, the architecture in cell blocks facilitates diagnosis. Further, cell blocks increase diagnostic yield when combined with other preparations 1,2 by making available additional or otherwise remaining unused samples. When adequately cellular, cell blocks serve as a source of multiple additional sections that are valuable in the evaluation of more cells and for performing ancillary studies, such as special staining, immunostaining, ultrastructural analysis, and molecular testing. Most ancillary testing platforms are developed on formalin-fixed paraffin-embedded (FFPE) tissue. So cell blocks processed in this way generally do not require additional validation, and when performing immunostaining, the same slides serve as controls for surgical specimens and cell blocks. 8 Cell blocks, like histology blocks, can be archived and provide tissue for future diagnostic and research purposes, thereby preserving the original smears. 9 Nontraditional and future applications for cell blocks include microarray construction 10 and the ability to scan images and review them remotely without using Z-function, which entails additional resources (eg, time and storage). 11 In contrast to H&E cell block slides, LBC and smears have 3-dimensionality, where the z-axis may be greater than 30 lm in area; capturing all elements thus requires 3-D multiplane technology, also referred to as Z-function Arch Pathol Lab Med Vol 140, December 2016 Cell Blocks Saqi

2 CELL BLOCKS: METHODS Cell block preparation among laboratories is variable, but usually a common thread runs through these methods concentration of cells (generally with a centrifugation step), removal of the supernatant, formation of a cohesive pellet (often with a binding agent eg, plasma/thrombin and HistoGel [Thermo Scientific Richard Allan Scientific, Waltham, Massachusetts]), and processing of the pellet in histology like a tissue sample. Currently, there is no standardized practice for generating cell blocks, but plasma thrombin and HistoGel are the most commonly used methods. 12 In the former, plasma and thrombin are added to the cells to facilitate formation of a cohesive pellet. Data suggest that this technique is more effective when the specimen is collected in nonfixative solutions (eg, saline); cell blocks are not as well formed in specimens fixed in formalin, as it cross-links and interferes with the clotting cascade. 13 Disadvantages of this technique include an inability to control clot formation and irregular distribution of cells within the pellet, but reportedly these can be overcome by constant agitation. 8 Also, caution has to be exercised when selecting thrombin, as some thromboplastin agents contain epithelial cells that may interfere not only with diagnosis but also with interpretation of ancillary studies. 14 Meanwhile, the HistoGel technique uses an agarose base to congeal the cell pellet. This method requires heating of the HistoGel to a liquid state, adding it to the pellet, and cooling the mixture to solidify it before placing it in formalin for histology processing. Meticulousness is required to assess the amount of gel necessary adding too little prevents cohesion, while excess dilutes the specimen. Variations on the abovementioned techniques include tissue coagulum and collodion methods. In the former, an FNA sample, typically admixed with blood, is expelled onto filter paper and air-dried; following this, the coagulum is placed in formalin for histology processing. 15 For the collodion bag technique, collodion, a liquid polymer nitrocellulose material (Cardinal Health Inc, Dublin, Ohio), is poured into a tube and then removed when it has completely coated the walls. Once the collodion sets in the tube, it forms a bag in the shape of the tube into which a specimen is placed to capture the cells. Following centrifugation, the bag is removed from the tube, secured with a string, and placed in a cassette for tissue processing. As collodion is admixed with an ether-based solvent, it has to be handled in a laminar flow hood and stored in small volumes in a flameproof enclosure. 13 All of the aforementioned techniques require technical skills, which may not be uniform across individuals and laboratories, and more importantly, produce inconsistent results principally low cellular yield. 12 A recent study that compared cell blocks from benchtop FNAs, performed from surgical resections by using 3 techniques (plasma thrombin, HistoGel, and collodion bag), showed significantly greater cellular yield with collodion. 13 A few factors likely contributed to its success: this method captures all cells, it does not dilute the specimen with additional agent(s), and it is prepared by technicians skilled in the methodology. The Cellient system (Hologic), an automated cell block technology built on the ThinPrep system, uses vacuumassisted filtration to capture and concentrate available cells. Reported results vary from showing benefit to no significant difference in cellularity when compared with one of the more traditional methods. 12,16,17 Possibly, this may result because only an aliquot rather than all of the available material is used. 13 Moreover, specimens processed in Cellient are typically collected in a methanol-based solution, which therefore requires laboratory validation to ensure that the results of immunostains correspond to those achieved with formalin fixation. Weaker immunostaining with some antibodies (eg, thyroid transcription factor-1 and p63) has been reported in cell blocks prepared with Cellient, 16 with methanol fixation (recommended by the manufacturer) being the potential culprit. In a recent study, however, specimens were prefixed in formalin for 30 or 60 minutes and washed with CytoLyt (Hologic) before standard methanol fixation in PreservCyt (Hologic) and processing in Cellient to achieve results similar to the traditionally prepared formalin-fixed cell blocks. 17 Further study of this methodology is necessary to investigate the results of additional antibodies (only select antibodies cytokeratin 7, AE1/AE3, CD45, and Melan-A were tested) and the manufacturer s recommendations. CELL BLOCKS: FIXATION Just as there is no consistent cell block preparation method, there is no standardized collection media. More common media include saline, Roswell Park Memorial Institute (RPMI) preservative, formalin, and alcohol-based media (ie, either methanol in CytoLyt 18 or methanol, ethanol, and isopropanol in SurePath 19 ), and each has its strengths and weaknesses. A cell block can be prepared from a specimen placed in any of the above, but these may not be suitable for all forms of ancillary testing, if necessary. A sample in saline provides flexibility to submit for cultures, flow cytometry, and LBC, but cell integrity is compromised, even over limited periods. A sample processed within 1 to 24 hours following collection in saline negatively and significantly impacts preservation, thereby compromising architectural and cytologic detail (eg, lyses the cells), possibly because saline is not perfectly isotonic with the cell cytoplasm. 13 RPMI is ideal for supporting cultures and leukocytes, especially when considering a (suspected) lymphoproliferative disorder, but it is not readily available at point-of-collection, partly because it needs to be refrigerated. Formalin is widely used and is the fixative on which many laboratory tests, such as immunohistochemistry and molecular analysis, are validated. Nonetheless, it may negatively impact amount of extractable DNA 20 and lead to chemical cross-links and DNA fragmentation that interfere with analysis. 9 As many laboratories have adopted LBC, specimens are often collected in one of the alcohol-based media; confirmation to ensure consistency across results, especially immunohistochemistry, is needed. Alcohol is at least comparable, if not better, than formalin for molecular testing. 9 A combination of alcohol and formalin 21 has also been used. Any of the above media are suitable for molecular testing. Per guidelines outlined by the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology, however, other fixatives and solutions (eg, Zenker, B5, B plus, acid zinc formalin, Bouin solution, decalcification) should not be used 9 for EGFR and ALK testing of lung carcinoma. CELL BLOCKS: DISADVANTAGES Despite their utility, cell blocks have disadvantages. Above all, they are not always sufficient, 8,22 with suboptimal cellularity reported in up to 57% of aspirate needle rinses. 12,23 Several key reasons may contribute to this. For Arch Pathol Lab Med Vol 140, December 2016 Cell Blocks Saqi 1319

3 instance, the nature of the lesion impacts cellular yield, as spindle and fibrotic processes tend to forfeit fewer cells. The availability of rapid on-site evaluation (ROSE) has downstream effects on cell block quality. With ROSE, the cytologist can not only request additional and dedicated material for a cell block but also evaluate for viability and contamination/dilution by nonlesional content, such as native epithelium and mucus. Even with a rich sample, 1 or more preanalytic variables that revolve mainly around a lack of standardization in best practices may negatively impact cell block cellularity. 24 Too often greater attention is focused on the diagnosis and not on procurement and triage, which have been shown to significantly affect cellularity and decrease the insufficient rate of molecular testing. 25 Sung et al 25 demonstrated that lack of adequate triage beginning at the time of tissue acquisition results in insufficient tissue on cell blocks for molecular testing of lung adenocarcinomas in up to 26% of endobronchial ultrasound-guided FNAs; with appropriate triage, only 4% of cases had insufficient tissue. 25 These results emphasize the significance of procurement, triage, and ROSE. Suboptimal cell blocks may result from poor specimen allocation making numerous thick smears with clots and tissue fragments that span the entire slide surface. At times, some of these scenarios are unavoidable owing to the nature of the specimen. Gently lifting the clots off the smears with a needle, or scraping excess material with a second slide and placing it in media are solutions to optimize cell blocks. Thick and blood-laden preparations hinder interpretation of cytologic details on smears; simultaneously, most of the cells and tissue fragments are present on the smears, leaving scant material for the cell blocks. Thinly smearing a few select tissue particles and allocating the remainder for a cell block, 26 in contrast, would optimize both smear and cell block preparations. Next, there are several ways to process cell blocks, most of which are quite skill-dependent 8 and labor-intensive. While the cost of supplies for these nonautomated methods is relatively minimal, the cost of labor is expected to be higher, and quality (ie, cellularity) may potentially be compromised in low-resource and/or high-volume settings. Under time constraints for instance, there are risks of (1) sample loss while separating the supernatant from the cells of interest, (2) dilution due to addition of excess congealing agent (eg, plasma/thrombin or HistoGel), and/or (3) incomplete pellet formation. Further, potential cellular loss in an already small specimen may occur during multiple transfers during processing (eg, handling and embedding in paraffin in the histology laboratory). Thus, all of these factors lead to inconsistencies among laboratories, with variable rates of success and failure with cell blocks, as described in the literature. 12 One currently available automated method, Cellient, addresses many of these deficiencies but it entails a significant capital purchase or lease and offers only serial (ie, only one block is processed at a time), rather than parallel (ie, multiple blocks simultaneously), processing with an average preparation time of 45 minutes per cell block. 11 CELL BLOCK ALTERNATIVES: SMEARS Smears, LBC, and/or cell blocks are typically prepared from FNAs and non-fnas (ie, exfoliative specimens), with the latter representing a much greater specimen volume. Besides cell blocks, ancillary testing can be performed on other preparations including LBC (eg, ThinPreps) and direct smears. 22,27,28 Of the two, only smears are discussed, since they are more commonly used for molecular testing and described in the recent literature. Especially in the absence of an adequate cell block, smears provide an invaluable alternative to a repeat and/or more invasive procedure. So why prepare cell blocks? How do cell blocks and smears measure up to each other? When ROSE is provided, direct smears are assessed for tumor content. To enrich diagnostic content, Knoepp el al 23 propose preparing 2 stained smears, 1 air-dried for Diff- Quik staining for ROSE and 1 alcohol fixed for subsequent Papanicolaou staining, and additional unstained slides. Examination of the slides both Diff-Quik smear and unfixed-unstained slides under the microscope offers an estimate of cellularity for potential ancillary testing. The composition of a cell block cannot be determined by using the same approach; nevertheless, gross examination of the specimen for firm, tan-white tissue particles and extrapolation of tumor content from a Diff-Quik smear, similar to that outlined by Knoepp et al, 23 function as indicators of adequacy. With both stained smears and cell blocks, the malignant cell content can be directly visualized to ensure adequate cellularity for molecular testing 9 ; such visible inspection is not possible when using residua of LBC, though it may be inferred from the corresponding LBC slide. Ancillary testing can be performed on direct smears, but additional slides have to be available so as not to sacrifice all of the diagnostic material. Options to circumvent this situation include slide scanning and lifting and transferring some cells off smears. Immunohistochemistry is generally validated on FFPE samples, so testing performed on direct smears and LBC has to be evaluated for false-negative and false-positive staining. At times, background staining may hinder interpretation of immunostain results of smears. Successful molecular testing off direct smears has been described, 22,29 and smears provide high-quality DNA. 29 In fact, smears are the primary or major source of material for molecular testing in some institutions. da Cunha Santos et al 24 performed an extensive analysis of the impact of preanalytic variables, including specimen type (ie, cell block, smears, and LBC), upon EGFR mutation testing of lung carcinomas. The results between smears and cell blocks were overall similar with reference to suitability for molecular testing and test failure rates. When compared with LBC, smears had greater DNA yield. 24 In a study designed to test for BRAF mutation in thyroid aspirates, LBC samples underwent degradation and lacked quality DNA for polymerase chain reaction analysis 9 months following processing. 30 Some observations may require clarification for instance, the optimal smear preparation for molecular testing. Killian et al 29 noted differences between air-dried, Diff-Quik stained, and alcohol-fixed, Papanicolaou-stained smears in molecular weight distribution of extracted DNA and increased degradation of the Papanicolaou-stained cells. The exact periods during which DNA from smears is stable and available for molecular testing is unknown, but there are reports of it being readily extractable from 6 months, months, 30 or up to 14 years 31 following acquisition. Potential drawbacks for archival smears include up to 48 hours for coverslip removal. 29 The slide type (eg, charged, fully frosted, and nonfrosted) can affect DNA yield as well. 32 Direct slide preparation is not labor-intensive but requires skill, though significantly less than that necessary for nonautomated cell block techniques, to prepare smears that 1320 Arch Pathol Lab Med Vol 140, December 2016 Cell Blocks Saqi

4 are small and thin, and without obscuring clots. The subsequent steps for ancillary testing on smears, nonetheless, can be time-intensive and/or skill-dependent: for instance, removing the coverslip if a stained/coverslipped slide is used or lifting and transferring tissue to create multiple slides for a broad panel of immunostains or molecular testing. Cutting multiple sections from a cell block, in contrast, is more efficient and less labor-intensive. As most molecular pathology laboratories are validated for FFPE samples, cell blocks integrate more seamlessly into the workflow, whereas incorporating smears into routine practice may potentially be more complex operationally. CELL BLOCKS: WHY SO MUCH FOCUS? CAN THEY BE IMPROVED? Cell blocks are not novel and their added value has been appreciated for decades. Why do they play a pivotal role now? Of all cytology preparations, they are most like surgical pathology specimens, including biopsies and resections. By mimicking the collection and fixation (ie, formalin) protocols of surgical pathology specimens, interference from other variables is controlled for and less likely to occur. In many laboratories, cell blocks may also assimilate more effortlessly into the existing workflow for ancillary testing. Moreover, expert consensus opinion recommends cell blocks over smears for EGFR mutation and ALK rearrangement evaluation in lung cancers. 9 An update on this topic is expected, however. 33 Broader molecular panels performed by commercial companies and laboratories also tend to be on FFPE samples. Moreover, the US Food and Drug Administration has approved ALK D5F3 (Cell Signaling Technology, Danvers, Massachusetts), an immunohistochemical stain typically performed on FFPE, as a companion diagnostic for ALK-directed therapy. Going forward, there is potential for other immunohistochemical antibodies to emerge from the pipeline, in which case FFPE cell blocks would obviate the need for vigorous validation. Currently, there is no single platform for immunohistochemical PD-L1 testing, and surgical pathology specimens are the tissue of choice. Again, if approved and used on cytology specimens, results of FFPE cell blocks would most closely resemble those of histology tissues. In the current state, however, cell blocks are not consistently optimal, with low cellularity being the leading cause for dissatisfaction. 12 Several steps can be undertaken to enhance cellularity. First, close collaboration between the pathologist and interventionalists (eg, radiologist, pulmonologist, endoscopist) to establish specimen processing protocols can improve outcomes. 34 There should be an initiative to share best practices collected from laboratories with high success rates. This is necessary, because even with a single method, there are variations in processing. For instance, HistoGel can simply be vortexed with the centrifuged sample, or a marker can be incorporated into the method to help the histotechnologist recognize the location of the cells in a block that may otherwise be scant and/or clear; when this is the case, the tissue is difficult to identify and potentially inadvertently exhausted or not sectioned at all. 35 Optimal tissue collection 22 (eg, additional dedicated passes) and triage (judicious allocation among smears, LBC, and cell blocks) are crucial 8,25,26,36 ; these increase the likelihood of sufficient sample for ancillary testing and preclude repeated procedure(s) 8 (Figure, A and B; Table). There are several factors that affect cellular yield in cell blocks. A, Low cellular yield. B, High cellular yield. Triage impacts cellularity. If excessive numbers of large smears containing clots and tissue fragments are made, the cell blocks will have low cellularity. Knowledge of tumor characteristics (eg, color of normal versus abnormal tissue) from the bench in the gross room can be applied at the time of ROSE to assess cellularity. Many tumors tend to be tan-white; thus, one can extrapolate that a diagnostic smear prepared from such particles suggests that other similar-appearing fragments are also lesional. Pitfalls to this method include interpreting native or fibrotic tissue as a diagnostic specimen. Separating diagnostic FNA passes from nondiagnostic (eg, those containing predominantly blood or necrosis) ones prevents specimen dilution. Several articles describe that when cell blocks fail, direct smears have sufficient cell content for molecular testing. This indicates that it is not necessarily the overall specimen cellularity that is suboptimal, but rather that other factors are at play. In such instances of sufficient cellularity in smears but not cell blocks, possibly most of the material is allocated to the smears; by redirecting some of the content from the smears, the yield of a cell block can be enhanced. If too much material is smeared, some can be scraped and Optimization of Cell Blocks Develop protocols for specimen collection. Provide rapid on-site evaluation. Triage specimen (allocate greater material for cell blocks over smears/lbc following diagnosis). Identify and modify shortcomings of cell block procedure in laboratory. Abbreviation: LBC, liquid-based cytology. Arch Pathol Lab Med Vol 140, December 2016 Cell Blocks Saqi 1321

5 placed in media for cell block at the time of ROSE. Others have even described using this technique after complete processing and coverslipping 37,38 ; the effect on ancillary testing requires further study. Variations in preparation and technical skill are the other likely sources of low-yield cell blocks. There are ongoing efforts to improve the existing techniques, and descriptions of new ones, 35,39,40 including a pilot study comparing HistoGel with a new methodology, with the latter demonstrating up to 7 times greater cellularity. 39 In summary, cell blocks are an integral part of cytology preparations and ancillary testing. In certain settings, such as molecular testing of lung cancer or by a commercial laboratory, they are the preferred cytology preparation. 9 To optimize them, care in specimen procurement, triage, and improvement in current processing techniques are necessary. 41 References 1. Calabretto ML, Giol L, Sulfaro S. Diagnostic utility of cell-block from bronchial washing in pulmonary neoplasms. Diagn Cytopathol. 1996;15(3): Collins GR, Thomas J, Joshi N, Zhang S. The diagnostic value of cell block as an adjunct to liquid-based cytology of bronchial washing specimens in the diagnosis and subclassification of pulmonary neoplasms. Cancer Cytopathol. 2012;120(2): Flint A. Detection of pulmonary neoplasms by bronchial washings: are cell blocks a diagnostic aid? Acta Cytol. 1993;37(1): Bahrenburg LPH. On the diagnostic results of the microscopical examination of the ascitic fluid in two cases of carcinoma involving the peritoneum. Cleveland Med Gaz. 1896;11: Chapman CB, Whalen EJ. The examination of serous fluids by the cellblock technic. N Engl J Med. 1947;237(7): Loukeris K, Vazquez MF, Sica G, et al. Cytological cell blocks: predictors of squamous cell carcinoma and adenocarcinoma subtypes. Diagn Cytopathol. 2012;40(5): Park GS, Lee SH, Jung SL, Jung CK. Liquid-based cytology in the fine needle aspiration of parathyroid lesions: a comparison study with the conventional smear, ThinPrep, and SurePath. Int J Clin Exp Pathol. 2015;8(10): Jain D, Mathur SR, Iyer VK. Cell blocks in cytopathology: a review of preparative methods, utility in diagnosis and role in ancillary studies. Cytopathology. 2014;25(6): Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Thorac Oncol. 2013;8(7): Wen CH, Su YC, Wang SL, Yang SF, Chai CY. Application of the microarray technique to cell blocks. Acta Cytol. 2007;51(1): Tawfik O, Davis M, Dillon S, et al. Whole-slide imaging of Pap cellblock preparations is a potentially valid screening method. Acta Cytol. 2015;59(2): Crapanzano JP, Heymann JJ, Monaco S, Nassar A, Saqi A. The state of cell block variation and satisfaction in the era of molecular diagnostics and personalized medicine. Cytojournal. 2014;11: Balassanian R, Wool GD, Ono JC, et al. A superior method for cell block preparation for fine-needle aspiration biopsies [published online ahead of print April 22, 2016]. Cancer Cytopathol. doi: /cncy Henwood AF, Charlton A. Extraneous epithelial cells from thromboplastin in cell blocks. Cytopathology. 2014;25(6): Yung RC, Otell S, Illei P, et al. Improvement of cellularity on cell block preparations using the so-called tissue coagulum clot method during endobronchial ultrasound-guided transbronchial fine-needle aspiration. Cancer Cytopathol. 2012;120(3): Wagner DG, Russell DK, Benson JM, Schneider AE, Hoda RS, Bonfiglio TA. Cellient automated cell block versus traditional cell block preparation: a comparison of morphologic features and immunohistochemical staining. Diagn Cytopathol. 2011;39(10): Prendeville S, Brosnan T, Browne TJ, McCarthy J. Automated Cellient() cytoblocks: better, stronger, faster? Cytopathology. 2014;25(6): Hologic Material Safety Data Sheet. ThinPrep CytoLyt Solution MSDS.pdf. Accessed March, BD Material Safety Data Sheet. data/attachments/0000/1125/surepath msds.pdf. Accessed March, Serth J, Kuczyk MA, Paeslack U, Lichtinghagen R, Jonas U. Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections. Am J Pathol. 2000;156(4): Nathan NA, Narayan E, Smith MM, Horn MJ. Cell block cytology: improved preparation and its efficacy in diagnostic cytology. Am J Clin Pathol. 2000;114(4): Knoepp SM, Roh MH. Reply to Ancillary techniques on direct-smear aspirate slides: a significant evolution for cytopathology techniques. Cancer Cytopathol. 2013;121(5): Knoepp SM, Roh MH. Ancillary techniques on direct-smear aspirate slides: a significant evolution for cytopathology techniques. Cancer Cytopathol. 2013; 121(3): da Cunha Santos G, Saieg MA. Preanalytic parameters in epidermal growth factor receptor mutation testing for non-small cell lung carcinoma: a review of cytologic series. Cancer Cytopathol. 2015;123(11): Sung SC, Crapanzano JC, DiBardino D, Swinarski D, Bulman WA, Saqi A. Molecular testing (MT) on endobronchial ultrasound (EBUS) fine needle aspirates (FNA): significance of appropriate specimen triage (ST) at the start of the procedure. Mod Pathol. 2016;29: Saqi A, Crapanzano JP. Optimization and triage of small specimens. In: Moreira A, Saqi A, eds. Diagnosing Non-small Cell Carcinoma in Small Biopsy and Cytology. New York, NY: Springer; 2015: Betz BL, Dixon CA, Weigelin HC, Knoepp SM, Roh MH. The use of stained cytologic direct smears for ALK gene rearrangement analysis of lung adenocarcinoma. Cancer Cytopathol. 2013;121(9): Betz BL, Roh MH, Weigelin HC, et al. The application of molecular diagnostic studies interrogating EGFR and KRAS mutations to stained cytologic smears of lung carcinoma. Am J Clin Pathol. 2011;136(4): Killian JK, Walker RL, Suuriniemi M, et al. Archival fine-needle aspiration cytopathology (FNAC) samples: untapped resource for clinical molecular profiling. J Mol Diagn. 2010;12(6): Kim WY, Oh SY, Kim H, Hwang TS. DNA degradation in liquid-based cytology and its comparison with conventional smear. Diagn Cytopathol. 2016; 44(5): Colanta A, Lin O, Tafe L, et al. BRAF mutation analysis of fine-needle aspiration biopsies of papillary thyroid carcinoma: impact on diagnosis and prognosis. Acta Cytol. 2011;55(6): Roy-Chowdhuri S, Goswami RS, Chen H, et al. Factors affecting the success of next-generation sequencing in cytology specimens. Cancer Cytopathol. 2015;123(11): Roy-Chowdhuri S, Aisner DL, Allen TC, et al. Biomarker testing in lung carcinoma cytology specimens: a perspective from members of the Pulmonary Pathology Society [published online ahead of print April 15, 2016]. Arch Pathol Lab Med. doi: /arpa sa. 34. Bulman W, Saqi A, Powell CA. Acquisition and processing of endobronchial ultrasound-guided transbronchial needle aspiration specimens in the era of targeted lung cancer chemotherapy. Am J Respir Crit Care Med. 2012;185(6): Varsegi GM, Shidham V. Cell block preparation from cytology specimen with predominance of individually scattered cells. J Vis Exp. 2009(29): Crapanzano JP, Saqi A. Adequacy and tissue preservation of small biopsy and cytology specimens. In: Moreira A, Saqi A, eds. Diagnosing Non-small Cell Carcinoma in Small Biopsy and Cytology. New York, NY: Springer; 2015: Kulkarni MB, Prabhudesai NM, Desai SB, Borges AM. Scrape cell-block technique for fine needle aspiration cytology smears. Cytopathology. 2000;11(3): Nga ME, Lim GL, Barbro N, Chan NH. Successful retrieval of fine-needle aspiration biopsy material from previously stained smears for immunocytochemistry: a novel technique applied to three soft tissue tumors. Mod Pathol. 2005; 18(5): Saqi A, Persaud R, Amoros C, Wu TT, Valladares-Silva S, Yeager K. New disposable cell block processing device and method for high cellular yield. JAm Soc Cytopathol. 2014;3(5):S79 S Balassanian R, Wool GD, Ono JC, et al. A superior technique for cell block preparation for fine needle aspiration. Mod Path. 2013;26(S2). 41. Harada S, Agosto-Arroyo E, Levesque JA, et al. Poor cell block adequacy rate for molecular testing improved with the addition of Diff-Quik-stained smears: need for better cell block processing. Cancer Cytopathol. 2015;123(8): Arch Pathol Lab Med Vol 140, December 2016 Cell Blocks Saqi